Salmonellae enterica are a genus of Gram-negative enteric bacteria that cause a range of human diseases, from gastroenteritis to typhoid fever and septicemia. Salmonella spp. present a significant global health burden, and development of new therapies to treat and prevent disease will require a detailed understanding of the molecular mechanisms underlying Salmonella pathogenesis. Salmonella is spread through contaminated food and water, and bacterial invasion of non-phagocytic intestinal epithelial cells is a key step in the establishment of infection. Salmonella bacteria induce their own uptake and survive within host cells by injecting 'effector' proteins that manipulate actin dynamics and host signaling directly into the host cell through a type III secretion system (T3SS). Coordination of multiple effectors and their targets is essential for efficient invasion, but the underlying mechanisms are not well understood. SipC is a core component of the T3SS that also functions as an effector. The studies outlined in this proposal test the hypothesis that SipC scaffolds the assembly of signaling complexes that promote bacterial invasion and intracellular survival. Previous work in the lab identified more than dozen SipC-interacting proteins through a yeast-two- hybrid screen, several of which directly bind or modulate actin. From this group, we have confirmed that filamin, FHOD1, and MAP4K4 associate with SipC in HeLa cell extracts. In Aim 1, their function in actin reorganization and bacterial uptake during infection will be characterized using RNAi in combination with immunofluorescence and bacterial internalization assays. In addition, we will explore the role of SipC in regulating their localization and activity through depletion/rescue assays using proteins deficient in binding to SipC. Salmonella infection induces activation of Akt kinase to promote intracellular bacterial growth. We have discovered that SipC binds directly to Akt and that the SipC interacting protein Exo70, a component of the exocyst complex, is essential for Salmonella-induced activation of Akt. Aim 2 describes experiments to characterize the roles of SipC and the exocyst complex in regulating Akt signaling. SipC mutants deficient in binding to Akt will be used in deletion/complementation experiments to determine how SipC regulates the localization, activation, and downstream signaling of Akt during infection. Co-precipitation and immunofluorescence assays will be used to determine whether depletion of Exo70 affects recruitment of Akt or its upstream kinases to sites of bacterial invasion and/or complex formation between Akt and upstream kinases. Collectively, these studies will define a novel role for SipC as a scaffold for the assembly of host proteins involved in actin reorganization and bacterial signaling during Salmonella infection. Importantly, these results are likely to have broad implications for pathogenesis of Gram-negative bacteria and will serve as a foundation for future research on bacterial pathogenesis.